5-(diisopropylamino)-2-[[4-(dimethylamino)phenyl]azo]-3-methyl-1,3,4-thiadiazolium methyl sulphate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Remarks:

preliminary study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1986/1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

The influence of Basic Blue 159 upon organogenesis and the subsequent outcome of pregnancy was assessed in sexually mature female rats of the CD strain.
For the purpose of this preliminary assessment, Basic Blue 159 was administered by gavage at dose levels of 5, 20, or 80 mg/kg body weight/day to groups of 6 pregnant female rats from Day 6 to Day 15 inclusive of gestation. Control animals received the vehicle, distilled water, throughout the same period.
All females were killed on Day 20 of gestation for examination of their uterine contents.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

5-(diisopropylamino)-2-[[4-(dimethylamino)phenyl]azo]-3-methyl-1,3,4-thiadiazolium trichlorozincate(1-)

EC Number:

298-265-2

EC Name:

5-(diisopropylamino)-2-[[4-(dimethylamino)phenyl]azo]-3-methyl-1,3,4-thiadiazolium trichlorozincate(1-)

Cas Number:

93783-70-1

Molecular formula:

C17H27N6S.Cl3Zn

IUPAC Name:

5-(diisopropylamino)-2-{[4-(dimethylamino)phenyl]diazenyl}-3-methyl-1,3,4-thiadiazol-3-ium trichlorozincate(1-)

Test material form:

solid: particulate/powder

Details on test material:

Basic Blue 159

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CD strain of Sprague Dawley origin

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River U.K. Limited, Margate, Kent, UK
- Age at study initiation: approximately 9-11 weeks; virgin females.
- Weight at study initiation: in weight range 197 g to 231 g.
- Housing: the animals were housed inside a barriered, limited access, rodent facility.
At various stages of the study the maximum number of rats per cage was:
Stage Number of rats Gage type
M F
Acclimatisation - 5 RC1
Mating 1 1 RB3 modified
Gestation - 1 RB3
- Diet: the rats were allowed free access to a commercially-available laboratory animal diet (Labsure Laboratory Animal Diet No. 1). The manufacturers (Labsure, Lavender Mill, Manea, Cambridgeshire, England) supplied a Certificate of analysis with every batch.
- Water: tap water from the local domestic supply was freely available to the animals via polythene bottles and chromium-plated sipper tube
- Acclimation period: 5 days for females, during which were unspected daily to check their physical condition
- Contaminantes: no contaminantes were reasonably expected to be present in water, diet or bedding at levels known to be capable of interfering with the progress and outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: valuated daily; target were 21°C (range 18 - 25 °C)
- Humidity: valuated daily; target were 55 % (range 40 - 79 %)
- Air changes: the animal room had its own supply of filtered air which was passed to the atmosphere without re-circulation, providing approximately 15 room air changes per hour.
- Photoperiod: 12 hrs dark and 12 hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Basic Blue 159 was prepared freshly each day as a solution in distilled water. Dosages were of the material as supplied.
Volume-dosage of 10 ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Information on the homogeneity of mixing, stability and concentration of the experimental compound in the vehicle was determined by the Sponsor. Samples of each concentration of the test mixtures were taken during the first and last weeks of treatment and analysed by the Sponsor for test chemical content.

Details on mating procedure:

Females were paired on a one-to-one basis with stock males of the same strain.
Each morning following pairing, the trays beneath the cages were checked for ejected copulation plugs and a vaginal smear was prepared from each female and examined for the presence of spermatozoa.
The day on which a sperm positive vaginal smear or at least three copulation plugs were found was designated Day 0 of gestation.

Duration of treatment / exposure:

Animals were dosed daily from Day 6 to day 15 of gestation inclusive.

Frequency of treatment:

once per day

Duration of test:

until Day 20 of gestation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

80 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

6 animals for each of the four groups

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

CLINICAL SIGNS and MORTALITY
All animals were examined daily throughout the study and any visible signs of reaction to treatment were recorded, with details of type, severity, time of onset and duration.

BODY WEIGHT
Females were weighed on days 0, 3, 6, to 16 inclusive, 18 and 20 gestation.

FOOD CONSUMPTION
Fodd consumption was recorded for the periods ending days 3, 6, 9, 12, 16, 18 and 20 of gestation.

WATER CONSUMPTION
Water consumption was recorded for the periods ending days 3, 6, 9, 12, 16, 18 and 20 of gestation.

POST-MORTEM EXAMINATIONS
Animals killed prematurely because of reaction to treatment were subjected to a thorough macroscopic examination of the visceral organs. Specimens of any abnormal tissue were retained.
On day 20 of gestation, the surviving females were killed by inhaled carbon dioxide for examination of their uterine contents. Each animal was first examined macroscopically for evidence of disease or adverse reaction to treatment and specimens of abnormal tissues were retained.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: yes
Examinations included:
- Number of corpora lutea: yes
- Number of implantations: yes
- Number of early resorption site: yes
- Number and distribution of live and dead foetuses in each uterine horn: yes
- Individual placental: yes

Fetal examinations:

- External examinations: yes of individual foetuses
- The neck and the thoracic and abdominal cavities of approximately two thirds of each litter were dissected and examined: yes
- Weight and sex of foetuses: yes

Statistics:

The smnall sample size precluded meaningful statistical evaluation. the biological significance of any suggestive inter-group differences was assessed by reference to control data previously recorded in these laboratories.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The general condition of females in Group 2 (5 mg/kg body weight/day) was comparable with that of the Controls. Females in .Group 3 (20 mg/kg body weight/day) showed excessive salivation after dosing, the first signs were recorded after the fourth dose and daily until the end of the treatment period. Blue staining around the mouth was noted for one animal. All Group 4 females (80 mg/kg body weight/day) exhibited excessive salivation post-dosing commencing after the second or third dose. Blue staining was noted around the mouths of the majority of the group. After the third dose, one female showed tremors, respiratory difficulties and became prone shortly after dosing. Subsequently, four females in this group, became lethargic after receiving 6, 7 (2 animals) or 8 doses and showed a variety of other signs including prone posture, shallow respiration, tremor and loss of the eye closure reflex.

Mortality:

mortality observed, treatment-related

Description (incidence):

At 80 mg/kg body weight/day, 5 of 6 females had to be killed for animal welfare reasons

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Bodyweight gain of Group 2 females (5 mg/kg body weight/day) was similar to that of the Controls. Animals in Group 3 (20 mg/kg body weight/day) showed an initial, transient effect upon bodyweight gain, but the subsequent rate of weight gain was comparable with that of the Controls. The single Group 4 female (80 mg/kg body weight/day) that survived to term showed a depressed bodyweight gain during the majority of the dosing period. Following completion of treatment the rate of weight gain was comparable with that of the Controls.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food intake of the Group 2 females (5 mg/kg body weight/day) was unaffected by treatment. In Group 3 (20 mg/kg body weight/day) a marginal depression was recorded between Days 12 and 15 of gestation, but subsequently recovered to control values. The one surviving Group 4 female (80 mg/kg body weight/day) showed a depressed food intake from the commencement of treatment and did not return to control values until Day 18 of gestation.

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Slight intergroup differences were recorded in the water intake of females in Groups 2 and 3 (5 and 20 mg/kg body weight/day), but there were no consistent effects attributable to treatment. Intake by the one female in Group 4 (80 mg/kg body weight/day) was depressed during Days 9 to 15 of gestation, but subsequently recovered.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Necropsy of surviving females at Day 20 of gestation revealed two Group 3 females (20 mg/kg body weight/day) with blue-stained salivary glands. No other treatment-related macroscopic abnormalities were observed.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

The one animal in Group 4 (80 mg/kg body weight/day) showed an increased pre-implantation loss

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

clinical signs

mortality

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

80 mg/kg body weight/day: depressed foetal and placental weights

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

It was concluded from this preliminary investigation that there were no indications to a teratogenic effect of Basic Blue 159. For the main study the highest dose should not exceed 20 mg/kg body weight/day.

Executive summary:

The influence of Basic Blue 159 upon organogenesis and the subsequent outcome of pregnancy was assessed in sexually mature female rats of the CD strain. For the purpose of this preliminary assessment, Basic Blue 159 was administered by gavage at dose levels of 5, 20, or 80 mg/kg body weight/day to groups of 6 pregnant female rats from Day 6 to Day 15 inclusive of gestation. Control animals received the vehicle, distilled water, throughout the same period. All females were killed on Day 20 of gestation for examination of their uterine contents.

Females receiving 5 mg/kg body weight/day showed no adverse response to treatment. At 20 and 80 mg/kg body weight/day all females exhibited excessive salivation after dosing. Five females receiving 80 mg/kg body weight/day were terminated before the end of treatment because of marked adverse responses, which included prone posture, lethargy, tremor, irregular respiration and loss of the eye closure reflex. At necropsy three of these females were found to have ulcerated areas in the fundic region of the stomach.

Weight gain of females receiving 5 mg/kg body weight/day was comparable with that of the control group. Females receiving

20 mg/kg body weight/day showed an initial, transient, reduction of weight gain, the subsequent rate of gain was comparable with that of the Controls. Weight gain of the single female that survived at 80 mg/kg body weight/day was depressed during the majority of the treatment period, but subsequeritly improved to the control rate of gain post treatment.

Food intake of females receiving 5 mg/kg body weight/day was unaffected by treatment. Females receiving 20 mg/kg body weight/day showed a marginal depression during the last four days of treatment, but subsequently recovered to control values. At 80 mg/kg body weight/day, food intake of the one surviving animal was reduced from the start of the treatment period.

Water intake of females receiving 5 and 20 mg/kg body weight/day was unaffected by treatment. At 80 mg/kg body weight/day, water intake was depressed during Days 9 to 15 of gestation, but subsequently improved to control values.

Litter responses of females receiving 5 and 20 mg/kg body weight/day were unaffected by treatment. Mean foetal and placental weight was reduced and pre-implantation loss was increased in the single surviving litter at 80 mg/kg body weight/day.

It was concluded from this preliminary investigation that there were no indications to a teratogenic effect of Basic Blue 159. For the main study the highest dose should not exceed 20 mg/kg body weight/day.